qPCR problems with chIP samples - (Dec/11/2008 )
Hi. I'm new to this forum, so I'm trying to figure out how to post and such. I've been trying to do chIP assays. I've been running into problems with getting a consistent standard curve for the dilutions of Input, so there is no way to actually analyze my IP samples and compare them to something. For the standard curves, I get ct values that sometimes increase, then decrease as I dilute the samples further (for example a 1:5 dilution will give a ct of 28, the 1:25 will give ct of 31, the 1:125 dilution gives a ct of 29). Sometimes the amount of sample doesn't even show up on the PCR, yielding a N/A value as an end read-out. The slopes of the standard curves are terrible and the correlation coefficients are really bad. I've tried multiple sets of primers at 100 nM final concentration in the PCR reaction, and nothing I'm doing seems to work, so I don't think that all of the primers I've designed can be totally bad. The melt curves look alright, no multiple products or anything. I've tried running a few different cycling protocols, but to no avail. I'm using the Biorad MyiQ cycler with the Biorad Sybr green master mix. Any ideas as to why this is happening???
HI there 
How do you purify your samples after ChIP?
Clare
I use a Qiagen PCR cleanup kit to purify the samples.
How do you purify your samples after ChIP?
Clare